Simultaneous determination of inflammatory factors SAA and LTF based on stable element labeling and inductively coupled plasma mass spectrometry to aid in the diagnosis of infection.

OBJECTIVE: The clinical value of Serum amyloid A (SAA) and Lactoferrin (LTF) has received significant attention, but their detection methods are inadequate, which limits their application. This study aims to develop a dual detection method based on stable element labeling strategies and inductively coupled plasma mass spectrometry (ICP-MS) for SAA/LTF and to assess whether it can be widely used in clinical practice. METHODS: A duplex immunoassay system based on sandwich method was constructed. After optimization, methodological evaluation was performed with the guidelines of Clinical Laboratory Standards Institute (CLSI). Finally, 131 plasma samples were collected to analyze whether the new method is suitable for clinical detection. RESULTS: The LoB, LLoQ, ULoQ, and linear range of the assay were 1.09 ng/mL, 3 ng/mL, 1500 ng/mL, 3-1500 ng/mL for SAA and 0.85 ng/mL, 2 ng/mL, 1200 ng/mL, 2-1200 ng/mL for LTF respectively. The recovery rates were 95.01% to 106.26%, the intra-batch precision of low, intermediate, and high-level samples was <8%, and the inter-batch of them was <11%, the deviation of interference test results was less than±10%. The Area Under the Curve (AUC) was 0.9809 for SAA, 0.8599 for LTF, and 0.9986 for combination. CONCLUSION: The quantitative duplex immunoassay for SAA/LTF has high accuracy, good precision, and high specificity, which meets the clinical testing requirements and can be widely used in clinical practice.

Identification of potential biomarkers for neuromyelitis optica by quantitative proteomics.

OBJECTIVE: Neuromyelitis optica (NMO) was a serious autoimmune inflammatory condition affecting the central nervous system. Currently, there was a lack of diagnostic biomarkers for AQP4-IgG-negative NMO patients. METHODS: A comparative proteomic analysis was conducted on the CSF of 10 patients with NMO and 10 patients with non-inflammatory neurological disorders (NND) using tandem mass tagging technology. Differentially expressed proteins (DEPs) were analyzed using bioinformatic methods. The candidate proteins were then validated through ELISAs in a subsequent cohort of 160 samples, consisting of paired CSF and plasma samples from 50 NMO patients, CSF samples from 30 NND patients, and plasma samples from 30 healthy individuals. RESULTS: We identified 389 proteins via proteomics, screening 79 DEPs. NCAM1, SST and AHSG were selected as candidate molecules for further validation. Compared to NND patients, there were decreased levels of AHSG in CSF and increased levels of NCAM1 and SST in NMO patients. The ELISA results revealed significantly higher levels of AHSG, SST and NCAM1 in the CSF of the NMO group compared to the NND group. Similarly, the serum levels of these three proteins were also higher in the NMO group compared to the healthy control group. It was found that serum NCAM1 levels significantly decreased in patients with non-relapsed NMO compared to patients with relapsed NMO and CSF NCAM1 level increased in patients with bilateral NMO compared to patients with unilateral NMO. Furthermore, CSF SST levels increased in AQP4 antibody-positive NMO patients compared to AQP4 antibody-negative patients. INTERPRETATION: CSF NCAM1, serum NCAM1 and serum SST may serve as potential biomarkers for NMO patients and aid in the diagnosis of AQP4 antibody-negative NMO patients.

Serum apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) is a novel stroke biomarker.

BACKGROUND: Apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) is a promising stroke biomarker. However, a large study of human serum ASC has not yet to be reported; additionally, the diagnostic value of prehospital concentration and practicality of ASC remains unknown. METHODS: We recruited 774 Chinese stroke patients, including 523 with ischemic stroke (IS) and 251 with hemorrhagic stroke (HS) within 14 days following symptom onset in the emergency department, alongside 481 healthy individuals and 64 cognitive impairment patients as controls. Serum ASC concentrations were determined using automated chemiluminescence immunoassay, exploring the relationship between serum ASC concentration and subtypes, severity, and sampling timepoints of stroke. RESULTS: ASC concentrations were significantly higher in stroke patients compared with all controls (P < 0.001). HS patients had greater ASC concentrations than IS patients (P < 0.05). With increasing ASC concentration, the proportion of severe cases increased. The area under the receiver operating characteristic curve (AUC) for differentiating between healthy individuals and stroke patients in the hyperacute phase was 0.78; this markedly improved (0.90) when considering samples from healthy individuals and patients with subarachnoid hemorrhage (SAH) ≤ 3  h from last-known-well (LKW). CONCLUSIONS: Serum ASC is a valuable biomarker for stroke differentiation and aids in the clinical diagnosis of stroke severity and subtypes.

The analysis of serum lipids profile in Guillain-Barre syndrome.

Background: Guillain-Barre syndrome (GBS) is an immune-mediated inflammatory peripheral neuropathy. This study aimed to conduct a systematic analysis of the serum lipids profile in GBS. Methods: We measured the serum lipids profile in 85 GBS patients and compared it with that of 85 healthy controls matched for age and sex. Additionally, we analyzed the correlation between lipids and the severity, subtypes, precursor infections, clinical outcomes, clinical symptoms, immunotherapy, and other laboratory markers of GBS. Results: Compared to the healthy controls, GBS exhibited significantly elevated levels of Apolipoprotein B (APOB), Apolipoprotein C2 (APOC2), Apolipoprotein C3 (APOC3), Apolipoprotein E (APOE), triglycerides (TG), and residual cholesterol (RC). Conversely, Apolipoprotein A1 (APOA1), Apolipoprotein A2 (APOA2), and high-density lipoprotein (HDL) were substantially lower in GBS. Severe GBS displayed noticeably higher levels of APOC3 and total cholesterol (TC) compared to those with mild disease. Regarding different clinical outcomes, readmitted GBS demonstrated higher RC expression than those who were not readmitted. Moreover, GBS who tested positive for neuro-virus antibody IGG in cerebrospinal fluid (CSF) exhibited heightened expression of APOC3 in comparison to those who tested negative. GBS with cranial nerve damage showed significantly reduced expression of HDL and APOA1 than those without such damage. Additionally, GBS experiencing limb pain demonstrated markedly decreased HDL expression. Patients showed a significant reduction in TC after intravenous immunoglobulin therapy. We observed a significant positive correlation between lipids and inflammatory markers, including TNF-α, IL-1ß, erythrocyte sedimentation rate (ESR), white blood cells, monocytes, and neutrophils in GBS. Notably, APOA1 exhibited a negative correlation with ESR. Furthermore, our findings suggest a potential association between lipids and the immune status of GBS. Conclusion: The research demonstrated a strong connection between lipids and the severity, subtypes, clinical outcomes, precursor infections, clinical symptoms, immunotherapy, inflammation, and immune status of GBS. This implies that a low-fat diet or the use of lipid-lowering medications may potentially serve as an approach for managing GBS, offering a fresh viewpoint for clinical treatment of this condition.

A retrospective study of myelin oligodendrocyte glycoprotein antibody-associated disease from a clinical laboratory perspective.

Objectives: To analyze the differences in laboratory data between patients with myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD), multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD). Methods: The study included 26 MOGAD patients who visited Beijing Tiantan Hospital from 2018 to 2021. MS and NMOSD patients who visited the clinic during the same period were selected as controls. Relevant indicators were compared between the MOGAD group and the MS/NMOSD groups, and the diagnostic performance of meaningful markers was assessed. Results: The MOGAD group showed a slight female preponderance of 57.7%, with an average onset age of 29.8 years. The absolute and relative counts of neutrophils were higher in the MOGAD group than in the MS group, while the proportion of lymphocytes was lower. The cerebrospinal fluid (CSF) IgG level, IgG index, 24-h IgG synthesis rate, and positive rate of oligoclonal bands (OCB) were lower in MOGAD patients than in the MS group. The area under ROC curve (AUC) was 0.939 when combining the relative lymphocyte count and IgG index. Compared to the NMOSD group, the MOGAD group had higher levels of serum complement C4 and lower levels of serum IgG. The AUC of serum C4 combined with FT4 was 0.783. Conclusion: Statistically significant markers were observed in the laboratory data of MOGAD patients compared to MS/NMOSD patients. The relative lymphocyte count combined with IgG index had excellent diagnostic efficacy for MOGAD and MS, while serum C4 combined with FT4 had better diagnostic efficacy for MOGAD and NMOSD.

Establishment of a comprehensive diagnostic model for neuromyelitis optica spectrum disorders based on the analysis of laboratory indicators and clinical data.

BACKGROUND: To establish a comprehensive diagnostic model for neuromyelitis optica spectrum disorders (NMOSDs) based on laboratory indicators and clinical data. METHODS: A retrospective method was used to query the medical records of patients with NMOSD from January 2019 to December 2021. At the same time, clinical data of other neurological diseases were also collected for comparison. Clinical data of the NMOSD group and non-NMOSD group were analyzed, and the diagnostic model was established based on these data. In addition, the model was evaluated and verified by the receiver operating curve. RESULTS: A total of 73 patients with NMOSD were included, and the ratio of males to females was 1:3.06. The indicators that showed differences between the NMOSD group and non NMOSD group included neutrophils (P = 0.0438), PT (P = 0.0028), APTT (P < 0.0001), CK (P = 0.002), IBIL (P = 0.0181), DBIL (P < 0.0001), TG (P = 0.0078), TC (P = 0.0117), LDL-C (P = 0.0054), ApoA1 (P = 0.0123), ApoB (P = 0.0217), TPO antibody (P = 0.012), T3 (P = 0.0446), B lymphocyte subsets (P = 0.0437), urine sg (P = 0.0123), urine pH (P = 0.0462), anti-SS-A antibody (P = 0.0036), RO-52 (P = 0.0138), CSF simplex virus antibody I-IGG (P = 0.0103), anti-AQP4 antibody (P < 0.0001), and anti-MOG antibody (P = 0.0036). Logistic regression analysis showed that changes in ocular symptoms, anti-SSA antibody, anti-TPO antibody, B lymphocyte subsets, anti-AQP4 antibody, anti-MOG antibody, TG, LDL, ApoB, and APTT had a significant impact on diagnosis. The AUC of the combined analysis was 0.959. The AUC of the new ROC for AQP4- and MOG- antibody negative NMOSD was 0.862. CONCLUSIONS: A diagnostic model was successfully established, which can play an important role in differential diagnosis of NMOSD.

Observational study of antibody levels after second and third SARS-CoV-2 vaccinations in medical workers.

BACKGROUND: Countries around the world are actively promoting vaccination against COVID-19. We observed the changes in serum neutralizing antibody titers in medical workers vaccinated with inactivated COVID-19 vaccine, in order to explore the necessity of a third dose of vaccination. METHODS: A total of 62 medical workers in our hospital were observed. Novel coronavirus neutralizing antibody titers in serum were detected by ELISA (enzyme-linked immunoassay). Neutralizing antibody tests followed in four batches according to the different time periods after three vaccinations. Sixty-two observers participated in the first batch of testing for neutralizing antibody, and 18 of them participated in all four batches. Fasting venous blood was taken from all the participants in the morning to detect serum neutralizing antibody titers. RESULTS: Sixty-two medical workers were divided into age groups of 21-30, 31-40, and >40 years, and the antibody titer in the oldest group was significantly lower than that in youngest group (p = 0.0137). There was a gradual decrease in antibody titers over time at around 1, 3, and 6 months after the second dose of vaccine (p < 0.0001). The antibody positive rate also decreased gradually (p = 0.0003). The neutralizing antibody titer around 1 month after the third dose was significantly increased (p < 0.0001). Unexpectedly, three participants with negative neutralizing antibody after the first and second dose produced neutralizing antibody with a measurable titer after the third dose. CONCLUSIONS: The neutralizing antibody titer in serum increased significantly after the third dose of vaccine. A third immunization even produced neutralizing antibody in previously negative individuals.

Establishment of SARS-CoV-2 Immunoglobulins (IgM, IgG) Reference Intervals for Elder Population in China based on 3,733 Samples.

BACKGROUND: Establishment of reference intervals (RIs) for different biomarkers is essential for clinical monitoring. The purpose of this study was to establish laboratory RIs of SARS-CoV-2 IgM and IgG for elder population. MATERIALS: Performance verification was conducted with reference to the Clinical and Laboratory Standards Institute (CLSI) guidelines, including linearity, imprecision, and allowable dilution ratio. Based on CLSI C28-A3 document, a total of 3,734 serum samples were collected, and 3,733 serum samples were used for the establishment of RIs for SARS-CoV-2 IgM and IgG. The subjects were grouped by gender and age. The age groups were as follows: 60 - 69 years, 70 - 79 years, 80 - 89 years, and 90 - 101 years. The RI was defined by nonparametric 95th percentile intervals. RESULTS: Percentage deviation of all the seven dilutions were all less than 12.5% during linearity evaluation. The inter-assay and intra-assay imprecision were all less than 5%. There is no significant difference between different gender and age groups for IgM (p = 0.0818, p = 0.7094), and there is significant difference between different gender and age groups for IgG (p = 0.0011, p = 0.0013). Harris-Boyd's test did not indicate partitioning for IgM and IgG. Cutoff values of RI for SARS-CoV-2 IgM and IgG were defined as 0.1523 S/CO and 0.2663 S/CO, respectively. CONCLUSIONS: RIs of SRAR-CoV-2 IgM and IgG were established for elder population, which can play an important role in the prevention and control of the epidemic.

Dual-detection fluorescent immunochromatographic assay for quantitative detection of SARS-CoV-2 spike RBD-ACE2 blocking neutralizing antibody.

The global effort against the COVID-19 pandemic dictates that routine quantitative detection of SARS-CoV-2 neutralizing antibodies is vital for assessing immunity following periodic revaccination against new viral variants. Here, we report a dual-detection fluorescent immunochromatographic assay (DFIA), with a built-in self-calibration process, that enables rapid quantitative detection of neutralizing antibodies that block binding between the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the angiotensin-converting enzyme 2 (ACE2). Thus, this assay is based on the inhibition of binding between ACE2 and the RBD of the SARS-CoV-2 spike protein by neutralizing antibodies, and the affinity of anti-human immunoglobulins for these neutralizing antibodies. Our self-calibrating DFIA shows improved precision and sensitivity with a wider dynamic linear range, due to the incorporation of a ratiometric algorithm of two-reverse linkage signals responding to an analyte. This was evident by the fact that no positive results (0/14) were observed in verified negative samples, while 22 positives were detected in 23 samples from verified convalescent plasma. A comparative analysis of the ability to detect neutralizing antibodies in 266 clinical serum samples including those from vaccine recipients, indicated that the overall percent agreement between DFIA and the commercial ELISA kit was 90.98%. Thus, the proposed DFIA provides a more reliable and accurate rapid test for detecting SARS-CoV-2 infections and vaccinations in the community. Therefore, the DFIA based strategy for detecting biomarkers, which uses a ratiometric algorithm based on affinity and inhibition reactions, may be applied to improve the performance of immunochromatographic assays.

ITIH4, as an inflammation biomarker, mainly increases in bacterial bloodstream infection.

Bloodstream infection (BSI) is usually accompanied with the changes of varieties of inflammation proteins. In our previous study, we identified that inter-α-trypsin inhibitor heavy chain H4 (ITIH4) was highly expressed in the infection arms than the normal control arm. However, the correlated verification and mechanism remain obscure. Escherichia coli infected mice model and clinical serum samples were used to validate the concentration of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), as well as ITIH4, in ELISA method. Cytokines (IL-6, TNF-α, IL-10 and lipopolysaccharide (LPS)) were used to stimulate the HepG2 cell model to explore which cytokines influence the expression of ITIH4. JAK/STAT inhibitor was treated before IL-6 and LPS stimulation. Westernblot, as well as real-time PCR were performed to detect the expression of ITIH4 in liver tissue from protein and transcription levels. Immunohistochemistry analysis was used to observe the expression of ITIH4 in mice liver tissue. In mice model, IL-6, TNF-α, as well as IL-10 increased in the infection arms than the normal control arm. ITIH4 in serum and liver tissue of mice model increased from 1 h to 128 h, which were remarkably different from that of the normal control arm. Besides, ITIH4 increased in the bacterial infection arm greatly than the fungemia arm, mycoplasma pneumoniae (MP) arm and febrile arm in clinical serum samples. Furthermore, using the HepG2 cell line, we demonstrated that ITIH4 was up-regulated at both protein and mRNA levels upon dose- and time- response treatments with IL-6, as well as LPS. Moreover, IL-6 or LPS mediated induction of ITIH4 expression could be significantly decreased by treatment with an JAK/STAT inhibitor in protein or mRNA level. No changes were observed after TNF-α or IL-10 stimulation. ITIH4 might be a critical inflammatory biomarker which correlated with the development of BSI, especially with bacterial bloodstream infection. It is expected that this study would provide some insights into potential functional mechanisms underlying BSI.

Evaluation of an Element-Tagged Duplex Immunoassay Coupled with Inductively Coupled Plasma Mass Spectrometry Detection: A Further Study for the Application of the New Assay in Clinical Laboratory.

BACKGROUND: Element-tagged immunoassay coupled with inductively coupled plasma mass spectrometry (ICP-MS) detection has the potential to revolutionize immunoassay analysis for multiplex detection. However, a further study referring to the standard evaluation and clinical sample verification is needed to ensure its reliability for simultaneous analysis in clinical laboratories. METHODS: Carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) were chosen for the duplex immunoassay. The performance of the assay was evaluated according to guidelines from the Clinical and Laboratory Standards Institute (CLSI). Moreover, reference intervals (RIs) of CEA and AFP were established. At last, 329 clinical samples were analyzed by the proposed method and results were compared with those obtained with electrochemiluminescent immunoassay (ECLIA) method. RESULTS: The measurement range of the assay was 2-940 ng/mL for CEA and 1.5-1000 ng/mL for AFP, with a detection limit of 0.94 ng/mL and 0.34 ng/mL, respectively. The inter-assay and intra-assay imprecision were all less than 6.58% and 10.62%, respectively. The RI of CEA and AFP was 0-3.84 ng/mL and 0-9.94 ng/mL, respectively. Regarding to clinical sample detection, no significant difference was observed between the proposed duplex assay and the ECLIA method. CONCLUSIONS: The ICP-MS-based duplex immunoassay was successfully developed and the analytical performance fully proved clinical applicability. Well, this could be different with other analytes.

Development and Evaluation of a Combined Immunoassay Method Based on a Stable Isotope Tagging Strategy and Inductively Coupled Plasma Mass Spectrometry for Detecting Human Chorionic Gonadotropin.

BACKGROUND: The development of a combined immunoassay method, based on a stable isotope tagging strategy and inductively coupled plasma mass spectrometry (ICP-MS), has created options for quantitative bioanalysis. The aim of the study was to develop a combined immunoassay, featuring ICP-MS and a stable element labeling strategy, for the detection of human chorionic gonadotropin (HCG), and developed methodology applicable for clinical practice. METHODS: In accordance with guidelines published by the Clinical and Laboratory Standards Institute (CLSI), we developed our assay and then evaluated its analytical performance, including the limit of detection (LOD), the upper limit of quantification (ULoQ), linearity, precision, recovery, cross reactivity, and interference. Next, we collected 130 clinical samples for analysis with the new assay. The data derived from our assay were then compared with those derived by an existing electrochemiluminescence immunoassay (ECLIA). RESULTS: The LOD of the assay was 0.33 mIU/mL and the ULoQ was 11,300 mIU/mL. The coefficient of determina-tion of linearity was higher than 0.99 in the range of 1 to 8,917 mIU/mL (R2 = 0.9964). The obtained recoveries ranged from 97.08% to 103.50%, while the intra-assay imprecision of high value samples and low value samples were 2.97% and 6.08%, respectively. The inter-assay imprecision of high value samples and low value samples were 3.98% and 7.08%, respectively. Interference test results deviated by less than ± 10% in the presence of hemoglobin ≤ 2 g/L, bilirubin ≤ 274 mol/L, or triglycerides ≤ 37 mmol/L. Compared with the commercial ECLIA method for clinical sample detection, the proposed method showed a significant correlation (R2 = 0.9770) and satisfactory agreement. CONCLUSIONS: The combination of ICP-MS and a stable element labeling based immunoassay for HCG detection was established successfully and the general performance of this system was acceptable, thus indicating that the assay has potential for the clinical application.

The COVID-19 pandemic.

In December 2019, an outbreak of pneumonia of unknown origin was reported in Wuhan, Hubei Province, China. Pneumonia cases were epidemiologically linked to the Huanan Seafood Wholesale Market. Inoculation of respiratory samples into human airway epithelial cells, Vero E6 and Huh7 cell lines, led to the isolation of a novel respiratory virus whose genome analysis showed it to be a novel coronavirus related to SARS-CoV, and therefore named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is a betacoronavirus belonging to the subgenus Sarbecovirus. The global spread of SARS-CoV-2 and the thousands of deaths caused by coronavirus disease (COVID-19) led the World Health Organization to declare a pandemic on 12 March 2020. To date, the world has paid a high toll in this pandemic in terms of human lives lost, economic repercussions and increased poverty. In this review, we provide information regarding the epidemiology, serological and molecular diagnosis, origin of SARS-CoV-2 and its ability to infect human cells, and safety issues. Then we focus on the available therapies to fight COVID-19, the development of vaccines, the role of artificial intelligence in the management of the pandemic and limiting the spread of the virus, the impact of the COVID-19 epidemic on our lifestyle, and preparation for a possible second wave.

Simultaneous determination of gastric cancer biomarkers pepsinogen PGI/PGII using element tagged immunoassay coupled with inductively coupled plasma mass spectrometry detection.

OBJECTIVES: In this study, a new immunoassay for the simultaneous determination of pepsinogen I (PGI) and pepsinogen II (PGII) in serum based on element labeling strategy coupled with inductively coupled plasma mass spectrometry (ICP-MS) detection was proposed. METHODS: The sandwich-type immunoassay was used to simultaneously detect PGI and PGII in serum. PGI and PGII were captured by anti-PGI and anti-PGII antibody immobilized on the magnetic beads and then banded with Eu3+ labeled anti-PGI detection antibody and Sm3+ labeled anti-PGII detection antibody, followed by ICP-MS detection. RESULTS: The linear correlation coefficient (R2 ) of PGI and PGII standard curves was .9938 and .9911, with the dynamic range of 0-200 ng/mL and 0-60 ng/mL, respectively. The limit of detection for PGI and PGII was 1.8 ng/mL and 0.3 ng/mL, respectively. The average recovery was 101.41% ± 6.74% for PGI and 101.47% ± 4.20% for PGII. Good correlations were obtained between the proposed method and CLIA (r = .9588 for PGI, r = .9853 for PGII). CONCLUSIONS: We established a mass spectrometry-based immunoassay for the simultaneous detection of PGI and PGII in a single analysis. The element tagged immunoassay coupled with ICP-MS detection has high sensitivity, accuracy, and specificity in clinical serum sample analysis.

A preliminary study for the establishment of a reference interval for vitamin B12 in China after performance verification of a second-generation ECLIA kit.

BACKGROUND: The second-generation electrochemiluminescence immunoassay (ECLIA) kit of vitamin B12 is widely used in clinical laboratories, and the establishment of a reference interval (RI) is essential to provide the basis for clinical monitoring. The purpose of this study was to establish a laboratory RI for vitamin B12 in China and at the same time verify the method performance of the second-generation kit. METHODS: The verification of the method performance was conducted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Based on these guidelines, a total of 580 serum samples were collected, and 391 serum samples were used for the establishment of the RI according to CLSI guidelines. The subjects were grouped by sex and age. The age groups were as follows: 21-40, 41-60, and 61-80 years. The RI was defined by nonparametric 2.5th and 97.5th percentile intervals. RESULTS: The performance of the second-generation kit of vitamin B12 from the Roche Cobas E602 system was in compliance with laboratory requirements. Serum vitamin B12 levels conformed to a non-Gaussian distribution. Harris-Boyd's test did not indicate partitioning for different age and gender group. Besides, there was no significant difference between different age groups (P = .07) and gender groups (P = .2002). The RI for healthy Chinese adults (aged 21-80 years) calculated by the nonparametric method was 250.8-957.1 pg/mL. CONCLUSIONS: The reference range of vitamin B12 was established, which provided a theoretical basis for the clinical application and monitoring of vitamin B12 detection.

Development and evaluation of an element-tagged immunoassay coupled with inductively coupled plasma mass spectrometry detection: can we apply the new assay in the clinical laboratory?

Introduction Element-tagged immunoassay coupled with inductively coupled plasma-mass spectrometry (ICP-MS) detection has the potential to revolutionize immunoassay analysis in clinical detection; however, a systematic evaluation with the standard guidelines of the assay is needed to ensure its performance meets the requirements of the clinical laboratory. Methods Carcinoembryonic antigen (CEA) was chosen for analysis using the proposed method. A systematic evaluation of the proposed assay was carried out according to the Clinical and Laboratory Standards Institute (CLSI). The 469 clinical samples were analyzed using the new method and compared with the electrochemiluminescent immunoassay (ECLIA) method. Results The measurement range of the assay was 1-900 ng/mL, with a detection limit of 0.83 ng/mL. The inter-assay and intra-assay imprecision were 4.67% and 5.38% with high concentration samples, and 9.27% and 17.64% with low concentration samples, respectively. The cross-reactivity (%) for different antigens was less than 0.05%, and the recovery was between 94% and 108%. Percentage deviation of all the dilutions was less than 12.5% during linearity estimation. The interference bias caused by different substances was less than 10%. The reference interval of the assay was 0-4.442 ng/mL. Comparison with the commercial ECLIA method for clinical sample detection, the proposed method showed a correlation of 0.9878 and no significant differences between the methods were observed (p = 0.6666). Conclusions The ICP-MS based immunoassay was successfully developed, and the analytical performance of the assay met the requirements of the CLSI, which fully proved the clinical transferability and application of the new method.

The Impact of Sharing Primer, the Quantity of the Internal Control Gene and the Primer Dimer on Reaction System in Duplex PCR.

BACKGROUND: In duplex real time quantitative PCR (qPCR), there are several factors affecting the sensitivity of duplex qPCR, such as sharing primer, quantity of the internal control (IC) gene, and the primer dimer (PD). The aim of the study is to find out the relationship of interference among templates with different primer pairs, the internal control gene, and the PD in duplex PCR. METHODS: We designed and synthesized plasmids with partial same sequence and different types of primers include central-homo primer pair, ordinary primer pair, and complementary primer pair. Then we compared the amplification efficiencies of different kinds of primer pairs. Besides, we adjusted the amount of IC plasmid and IC primer to find out the key factor that influences the sensitivity of the target template. RESULTS: The concentration ratios of two plasmids showed interference in sharing the universal primer pair, sharing one forward primer, and sharing no primer were 50:1, 200:1 and 500:1, respectively. The amplification efficiency of the ordinary primer pair was higher than that of the universal primer pair for the plasmid. Sensitivity of the duplex qPCR remained unchanged when the amount of PDs increased, but it declined rapidly when the quantity of the IC increased. CONCLUSIONS: IC is the major factor influencing the sensitivity of the duplex qPCR and it would be better to use one universal primer for IC and target template to achieve minimum interference.

Proteomic approaches in the discovery of potential urinary biomarkers of mucopolysaccharidosis type II.

Mucopolysaccharindosis type II (MPS II) is a rare lysosomal storage disorder caused by deficient or absent activity of the iduronate-2-sulfatase (IDS) enzyme, which leads to pathological accumulation of the glycosaminoglycans(GAGs). The absence of early diagnosis can result in irreversible developmental, neurological, and physiological damage. The lack of clear understanding of the etiology of physiological dysfunction in MPS II has been a major obstacle to the development of new treatment. Therefore, a reliable biomarker for early diagnosis and exploration of pathogenic mechanism are of great importance. Proteomics provides powerful tool for protein expression alterations and study of complicated pathological process. This study was performed to identify the differential protein profile in urine of MPS II patients using two-dimensional gel electrophoresis(2D-PAGE)combining with MALDI-TOF/TOF and a total of 15 differentially expressed proteins were identified. Content of alpha1-antitrypsin, Gm2 activator and lipocalin-type prostaglandin D synthase was measured by ELISA method. The value of urinary α1-AT/Cr in MPS II group was 0.79 ±â€¯0.10 mg/mmol, significantly higher than 0.42 ±â€¯0.05 mg/mmol in healthy control group; whereas the value of GM2A/Cr and L-PGDS/Cr in MPS II group was 1.30 ±â€¯0.12 µg/mmol and 9.86 ±â€¯1.16 ng/mmol respectively, which was significantly lower than 2.19 ±â€¯0.19 µg/mmol and 13.98 ±â€¯1.48 ng/mmol in healthy control group. The proteins can be considered as accessory diagnostic biomarkers for MPS II. This approach helped to discover early diagnostic markers and provided a better understanding of the pathogenic mechanism of MPS II.

Chemical-Modified Nucleotide-Based Elemental Tags for High-Sensitive Immunoassay.

Multiplex biomolecular analysis with inductively coupled plasma mass spectrometry (ICP-MS) becomes increasingly important in clinical diagnosis and single cell analysis. However, the sensitivity of ICP-MS-based immunoassay is only comparable or lower than those of fluorescence methods at the present stage. Therefore, designing elemental tags with a large number of metal atoms is necessary to achieve high-sensitive detection. In this work, we proposed a new strategy to build up elemental tag loading with hundreds of rare earth ions by coupling alkyne-DNA chains with rare earth element (REE)-DOTA complexes a click chemistry reaction. There are about 2 orders of magnitude improvement in sensitivity compared with single metal-ion tags. DNA chains with multialkynyl groups were facilely prepared by PCR synthesis. Moreover, the DNA-based elemental tags own excellent water-solubility and biocompatibility. The tags would be potentially applied to mass cytometry and clinical diagnosis.

New design of probe and central-homo primer pairs to improve TaqMan™ PCR accuracy for HBV detection.

Quantitative PCR (qPCR) assay using TaqMan™ probe was widely used in the detection of different nucleic acids. However, this technology has several drawbacks, including false negative results caused by primer-dimer (PD) and false positive issues due to primer-probe aggregations. Here, we designed a modified TaqMan™-Molecular Beacon probe by adding an antisense base and a new type of primer pair named central-homo primer pairs bearing 5-10 bases homologous sequence on the 3' end. Using the HBV qPCR assay as a proof of concept, the new design significantly improved the accuracy of the TaqMan™ qPCR assay for HBV detection. Application of the central-homo primer pair led to significantly delayed Ct values by 5-10 cycles compared with conventional primer design. The modified probe containing an antisense base did not produce any detectable signal in repeating primer-probe aggregation experiments. Furthermore, the use of the central-homo primer pair and the non-competitive internal control could solve the false negative problem caused by PD formation. We validated this customized duplex qPCR system using 208 clinical samples collected from patients in clinic showing accuracy was higher than that of the conventional qPCR method.